Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic transcriptional regulators. Following initial recruitment of PcG proteins to target loci, they are capable of maintaining transcriptional repression through an indefinite number of cell cycles. However, PcG-mediated repression is dynamic and can be reversed to allow the expression of target genes. Targets of mammalian PcG proteins include differentiation-specific genes in embryonic stem cells and pluripotency genes when stem cells are induced to differentiate. Mis-expression of PcG proteins contributes to a wide range of human cancers. Both repression of pluripotency genes and the oncogenic effects of PcG proteins involve de novo repression of previously active genes. Although much is known about the activities of PcG proteins during the maintenance phase of repression, virtually nothing is known about the mechanisms involved in the initial establishment of PcG-mediated repression or how PcG proteins initially distinguish between transcriptionally repressed and active targets. Recruitment and stable maintenance of Drosophila PcG proteins requires the presence of DNA sequences known as Polycomb Response Elements (PREs) that contain binding sites for multiple sequence-specific proteins. In mammals, noncoding RNAs and transcription factors have been proposed to recruit PcG proteins, although recent evidence suggests a higher correlation of the genomic distribution of PcG proteins with unmethylated CpG islands. It has been proposed that mammalian unmethylated CpG islands and Drosophila PREs may provide similar chromatin environments that are conducive to PcG protein targeting. In the proposed studies, we will use an established genetic system to produce embryos in which a PcG target gene is ubiquitously repressed. Embryos at discrete stages during the developmental window when PcG-mediated repression is established will be subjected to chromatin immunoprecipitation to identify the temporal recruitment of proteins and deposition of histone modifications. Two specific aims are proposed: (1) Determine the respective contributions of sequence-specific DNA binding proteins to the activities of a target gene's PREs; (2) Define the molecular and biochemical events that lead to establishment of PcG-mediated repression and the mechanism by which PcG proteins distinguish between repressed and active target genes. The results of these studies will be relevant to the establishment and manipulation of embryonic and induced pluripotent stem cells and to the mechanisms underlying the oncogenic activities of PcG proteins.